EP0707067A2 - Neues Gen für eine Thrombin-ähnliche Protease kodierend - Google Patents

Neues Gen für eine Thrombin-ähnliche Protease kodierend Download PDF

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Publication number
EP0707067A2
EP0707067A2 EP95305675A EP95305675A EP0707067A2 EP 0707067 A2 EP0707067 A2 EP 0707067A2 EP 95305675 A EP95305675 A EP 95305675A EP 95305675 A EP95305675 A EP 95305675A EP 0707067 A2 EP0707067 A2 EP 0707067A2
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European Patent Office
Prior art keywords
protease
amino acid
halybin
thrombin
sequence
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EP95305675A
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English (en)
French (fr)
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EP0707067A3 (de
Inventor
Hak-Dai Kim
Yung-Dae Yun
Kwang-Hoe Chung
Doo-Sik Kim
Hong-Mo Moon
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Mogam Biotechnology Research Institute
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Mogam Biotechnology Research Institute
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Publication of EP0707067A2 publication Critical patent/EP0707067A2/de
Publication of EP0707067A3 publication Critical patent/EP0707067A3/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel gene coding a protease, more specifically, a novel cDNA sequence of a thrombin-like serine protease originated from the venom gland of a Korean viper, Salmosa( Agkistrodon halys brevicaudus ) and an amino acid sequence translated therefrom.
  • thrombosis and hemostasis has been a principal subject in the medical field for long time.
  • studies on the thrombolytic agents such as urokinase, streptokinase and tissue-type plasminogen activator, have been actively carried out, and their use as therapeutic agents for thrombosis has been also practiced( see : Lijnen, H.R. et al., Thromb. Haemost., 66:88(1991)).
  • snake venoms which are known to influence the hemostatic system in human, has been also investigated, and a number of materials that have an effect on the hemostatic system have been isolated from the venom and clinically tested.
  • these materials many of which have been practically introduced in basic research or in diagnosis and treatment, in particular, a variety of thrombin-like proteases isolated from snake venoms which cleave fibrinopeptide of fibrinogen to produce fibrin, i.e., thrombin-like protease, have been reported( see : Meier, J. et al., Toxicology, 21:171(1991)).
  • 23 enzymes of this category have been identified and the amino acid sequences of four enzymes have been determined.
  • batroxobin a thrombin-like protease isolated from the venom of "Lance head snake” ( Bothrops atrox moojeni ), was reported to cleave fibrinopeptide A of fibrinogen in a specific manner.
  • a small amount of batroxobin is able to convert fibrinogen to fibrin I(Des-A fibrin) which is degraded by plasmin rapidly, thus resulting in a decrease of fibrinogen level in blood.
  • it can induce the synthesis of plasminogen activator and accelerate degradation of thrombus, but rather causes blood clotting when a high level of batroxobin is applied.
  • batroxobin has been developed both as a defibrinogenating drug (commercial name “Defibrase”) and as a hemostatic agent (commercial name “Reptilase”)( see : Meier, J. et al., Medical Use of Snake Venom proteins, CRC Press, 136(1990)).
  • the inventors isolated a cDNA of thrombin-like protease(hereinafter referred to as "Halybin”) from the cDNA library of the venom of Salmosa, and determined an amino acid sequence translated from the cDNA.
  • Halybin a cDNA of thrombin-like protease
  • a primary object of the present invention is, therefore, to provide a novel cDNA sequence of thrombin-like protease originated from the venom gland of a Korean viper, Salmosa( Agkistrodon halys brevicaudus ).
  • the other object of the invention is to provide an amino acid sequence of the thrombin-like protease translated from the cDNA sequence.
  • a cDNA of 2.1 kb encoding a thrombin-like protease was cloned, by employing an oligonucleotide probe for the non-conserved amino acids in the PCR products prepared with oligonucleotide primers for the amino acid sequences well conserved in the serine proteases isolated from the snake venom.
  • cDNA thus cloned was determined to contain an open reading frame of 837 nucleotides encoding 279 amino acids, where the deduced amino acid sequence was composed of a mature serine protease of 234 amino acids and a signal peptide of 45 amino acids.
  • Halybin was determined to have a molecular weight of 25.7 kDa and the isoelectric point(pI) of 5.68, and was suggested as a glycoprotein of higher molecular weight in the venom, since it contains an N-glycosylation site.
  • Halybin has 64-71 % homology in terms of amino acid sequence, in a relation to thrombin-like proteases known in the art; and, the amino acid sequences of the active site, e.g., His, Asp, and Ser and in the vicinity of the active site as well as in the N-terminal sequence, are well conserved, which is common phenomena in the serine proteases originated from the snake venom.
  • the protease of the present invention retains 12 cysteines, which are also well conserved throughout this category of enzymes, Halybin was suggested to have a thrombin-like protease activity similar to known proteases.
  • cDNA sequence of the Halybin originated from the venom of a Korean viper, Salmosa( Agkistrodon halys brevicaudus ), can be expressed in proper expression systems established in recombinant E. coli , yeast, baculovirus/insect cells and other animal cells.
  • the recombinant Halybin thus expressed can be applied as an active ingredient of thrombolytic and hemostatic agents.
  • Example 1 Construction of cDNA library from the venom gland of Salmosa
  • Venom gland was obtained from a Korean viper, Salmosa( Agkistrodon halys brevicaudus ). As the venom gland is a pea-like tiny tissue, at least five glands were used to obtain a sufficient amount of RNA. To isolate total cellular RNA, guanidine isothiocyanate was treated, and an ultracentrifugation with CsCl cushion was followed. Poly(A)+RNA was separated from the total cellular RNA, by applying on an oligo(dT)-cellulose column twice. Then, first and second strands of cDNA were synthesized by employing reverse transcriptase, RNase H, and E. coli DNA polymerase I( see : Sambrook, J.
  • cDNA library thus constructed was determined to contain 106 of independent plaques.
  • the construction process of the cDNA library from the venom gland of Salmosa was schematically depicted in Fig. 1.
  • oligonucleotide primers for the well conserved amino acids in serine proteases from the snake venom were first selected as follows: Based on the amino acid sequence common to the N-terminal region of serine proteases from the snake venom, i.e., VIGGDEC, a degenerated oligonucleotide containing the following sequence was employed as 5' primer: 5' GTIATIGGIGGNGA(T/C)GA(A/G)TG 3'(SEQ ID NO:13).
  • a degenerated oligonucleotide containing the following sequence was employed as 3' primer: 5' TGIGCIGCIGTNA(A/G)NCCCA 3'(SEQ ID NO:14) wherein, I represents inosine; and, N represents one of four nucleotides, i.e., A, G, C or T.
  • PCR polymerase chain reaction
  • the amino acid sequence derived from the DNA sequence (“PCR120") displayed 65-72 % homology with known serine proteases such as BATROXOBIN isolated from Bothrops atrox moojeni and FLAVOXOBIN isolated from Trimeresurus flavoviridis ( see : Fig. 2B).
  • the underlined and the boxed represent DNA sequence derived from the PCR primers and DNA sequence employed in the screening of cDNA library, and amino acid sequences translated therefrom, respectively; and, (*), ($) and (#) represent conserved amino acids, an amino acid residue constituting the active site among the conserved sequence and conserved cysteine residues, respectively.
  • the DNA sequence corresponding to the mid-domain of the PCR product of 120 bp i.e., 5'-CGTTCCCTTGTT- GTCTTGTTTAACTCCAGCG-3 ⁇ (SEQ ID NO:15), was employed as a probe for the screening of cDNA library described below.
  • cDNA library prepared in Example 1 was plated on E. coli XL-1 Blue to obtain plaques of 1.2 X 105. After transferring the plaques into a nitrocellulose membrane twice, the membrane was treated with a degenerating solution(0.5 N NaOH, 1.5 M NaCl) for 2 minutes, with a neutralizing solution(0.5 M Tris HCl(pH 7.4) containing 1.5 M NaCl) for 5 minutes, and washed in 2 X SSC for 30 seconds.
  • prehybridization was carried out at 68 °C for 3 hours, in a solution containing 5 X SSC, 20 mM sodium phosphate(pH 6.5), 3 % SDS and 100 ⁇ g/ml of salmon sperm DNA.
  • the probe selected in Example 2 was end-labelled with [ ⁇ -32P]ATP, added in a concentration of 1 X 106 cpm/ml to the prehybridization solution, and left to hybridize at 53 °C for 16 hours.
  • the membrane was washed with a solution containing 3 X SSC, 3 % SDS and 10 mM sodium phosphate(pH 6.5) at 53 °C for 1 hr.
  • the membrane was exposed to an X-ray film(Agfa curix, Germany) under an intensifying screen at -70 °C for 24 hours.
  • 11 positive clones whose spots were formed at the same position, were detected on the duplicate of the X-ray film.
  • Hybridization of phage plaques transferred from a plate to two separate sheets of nitrocellulose membrane and exposure on the X-ray film, reveals a positive result that spots are found at the same position on each membrane.
  • each clone was cloned in a pBluescript vector(Stratagene, USA) and treated with a restriction enzyme, and it was elucidated that the size of the insert ranged from 0.5 kb to 2.1 kb.
  • the sequence analysis of each clone proved that 11 clones share common sequence of a gene, and among them the full sequence of the longest clone of 2.1 kb was determined.
  • DNA sequence of 2.1 kb clone was determined by the dideoxy termination method( see : Sambrook, J. et al., Molecular Cloning, 2nd Ed., 1989) employing Sequenase(USB, USA). Careful determination of the sequence was carried out over a region of the total 2.1 kb of insert containing the total open reading frame. As a result, 1003 bp of DNA sequence was analyzed, which contains 5'-untranslated region of 49 bp, ORF(open reading frame) of 837 bp and 3'-untranslated region of 114 bp( see : Fig. 3A).
  • the cDNA contains an open reading frame of 837 nucleotides encoding 279 amino acids. 279 amino acids were also determined to contain a signal peptide of 45 amino acids and a mature enzyme of 234 amino acids( see : Fig. 3B). Presumed from the amino acid sequence and composition, the mature enzyme was determined to be a protein of molecular weight of 25.7 kDa with the isoelectric point(pI) of 5.68, and was suggested to be a glycoprotein of higher molecular weight in the venom, since it contains an N-glycosylation site.
  • Halybin displays 64-71 % homology in terms of amino acid sequence, in a relation to prior art thrombinlike proteases of snake venom( see : Fig. 5). Moreover, it was determined that Halybin contains amino acid sequences constituting the active site of common serine proteases, i.e., His, Asp, and Ser and the vicinity of the active site as well as the N-terminal sequence, which are well conserved in prior art serine proteases, e.g., FLAVOXOBIN( see : Shieh, T.-C. et al., J.
  • the present invention provides a cDNA sequence of a thrombin-like protease ("Halybin”) originated from the venom gland of a Korean viper, Salmosa ( Agkistrodon halys brevicaudus ), and an amino acid sequence translated therefrom.
  • the cDNA sequence of Halybin can be expressed in proper expression systems established in recombinant E. coli , yeast, baculovirus/insect cells and other animal cells, and Halybin thus produced can be applied as an active ingredient of thrombolytic and hemostatic agents.

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EP95305675A 1994-10-11 1995-08-15 Neues Gen für eine Thrombin-ähnliche Protease kodierend Withdrawn EP0707067A3 (de)

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KR9426001 1994-10-11
KR1019940026001A KR0127776B1 (ko) 1994-10-11 1994-10-11 신규한 단백질 분해효소의 cDNA 서열 및 그로 부터 유래된 단백질

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EP0707067A2 true EP0707067A2 (de) 1996-04-17
EP0707067A3 EP0707067A3 (de) 1997-01-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821106A (en) * 1996-06-21 1998-10-13 Mogam Biotechnology Research Institute CDNA of direct-acting fibrinolytic serine protease
WO2007046634A1 (en) * 2005-10-18 2007-04-26 Seoul National University Industry Foundation Method for preparing recombinant peptide from spider venom and analgesic composition containing the peptide

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4568545A (en) 1982-10-02 1986-02-04 Amano Seiyaku Kabushiki Kaisha Thrombolytic agent

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2517241B2 (ja) * 1986-08-19 1996-07-24 郁男 山科 遺伝子
JPH02124092A (ja) * 1988-11-04 1990-05-11 Fujisawa Pharmaceut Co Ltd バトロキソビンの遺伝子組換えによる製造法
DE4023699A1 (de) * 1990-07-26 1992-01-30 Basf Ag Neue proteine, ihre herstellung und verwendung

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4568545A (en) 1982-10-02 1986-02-04 Amano Seiyaku Kabushiki Kaisha Thrombolytic agent

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 108, 1988, Columbus, Ohio, US; abstract no. 90629N
H.K. GLOYD, PROC. BIOL. SOC., vol. 85, 1971, WASHINGTON, pages 577
H.R. LIJNEN ET AL., THROMB. HAEMOST., vol. 66, 1991, pages 88
J. MEIER ET AL., TOXICOLOGY, vol. 21, 1991, pages 171
J. MEIER ET AL.: "Medical Use of Snake Venom Proteins", 1990, CRC PRESS, pages: 136
J. SAMBROOK ET AL.: "Molecular Cloning", 1989, COLD SPRING HARBOR LABORATORY
J. SUN ET AL., ZHONGGUO YIKE DAXUE XUEBAO, vol. 16, 1987, pages 276
K.H. CHUNG ET AL., THROMB. HAEMOST. THHADQ, vol. 65, 1991, pages 953
K.Y. NAH ET AL., J. SURGERY, vol. 17, 1975, pages 13
M.W.C. HATTON, BIOCHEM. J., vol. 131, 1973, pages 799 - 807
N. ITOH ET AL., J. BIOL. CHEM., vol. 263, 1988, pages 7628 - 7631
R.T. SAWYER, BIO/TECHNOL., vol. 9, 1991, pages 513
S. NISHIDA ET AL., BIOCHEMISTRY, vol. 33, 1994, pages 1843 - 1849
S.J. GARDELL ET AL., J. BIOL. CHEM., vol. 264, 1989, pages 17947
T.-C. SHIEH ET AL., J. BIOCHEM., vol. 103, 1988, TOKYO, pages 696 - 605

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821106A (en) * 1996-06-21 1998-10-13 Mogam Biotechnology Research Institute CDNA of direct-acting fibrinolytic serine protease
WO2007046634A1 (en) * 2005-10-18 2007-04-26 Seoul National University Industry Foundation Method for preparing recombinant peptide from spider venom and analgesic composition containing the peptide

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EP0707067A3 (de) 1997-01-29
JPH08116980A (ja) 1996-05-14
KR0127776B1 (ko) 1997-12-29
KR960014345A (ko) 1996-05-22

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